The invention relates to the preparation and use of an isolated nucleic acid fragment which confers a Pi-ta resistance gene-mediated defense response in plants against disease caused by fungal pathogens. Genes incorporating such nucleic acid fragments either alone or in combination with an AVR-Pita isolated nucleic acid fragment or functionally equivalent subfragments thereof and suitable regulatory sequences can be used to create transgenic plants which can produce a Pi-ta resistance gene-mediated defense response against a variety of fungal pathogens, in particular, the rice blast fungus.
Plants can be damaged by a wide variety of pathogenic organisms including viruses, bacteria, fungi and nematodes. The invasion of a plant by a potential pathogen can result in a range of outcomes: the pathogen can successfully proliferate in the host, causing associated disease symptoms, or its growth can be halted by the host defenses. In some plant-pathogen interactions, the visible hallmark of an active defense response is the so-called hypersensitive response (HR). The HR involves rapid necrosis of cells near the site of the infection and may include the formation of a visible brown fleck or lesion. Pathogens which elicit an HR on a given host are said to be avirulent (AVR) on that host, the host is said to be resistant, and the plant-pathogen interaction is said to be incompatible. Strains which proliferate and cause disease on a particular host are said to be virulent, in this case the host is said to be susceptible, and the plant-pathogen interaction is said to be compatible.
Genetic analysis has been used to help elucidate the genetic basis of plant-pathogen recognition for those cases in which a series of strains (races) of a particular fungal or bacterial pathogen are either virulent or avirulent on a series of cultivars of a particular host species. In many such cases, genetic analysis of both the host and the pathogen revealed that many avirulent fungal and bacterial strains differ from virulent ones by the possession of one or more avirulence (xe2x80x9cavrxe2x80x9d or xe2x80x9cAVRxe2x80x9d) genes that have corresponding xe2x80x9cresistancexe2x80x9d (R) genes in the host.
This avirulence gene-resistance gene model is termed the xe2x80x9cgene-for-genexe2x80x9d model (Crute et al. (1985) pp 197-309 in: Mechanisms of Resistance of Plant Disease. R. S. S. Fraser, ed.; Ellingboe, (1981) Annu. Rev. Phytopathol. 19:125-143; Flor, (1971) Annu. Rev. Phythopathol. 9:275-296). According to a simple formulation of this model, plant resistance genes encode specific receptors for molecular signals generated by avr genes. Signal transduction pathway(s) then carry the signal to a set of target genes that initiate the host defenses. Despite this simple predictive model, the molecular basis of the avr-resistance gene interaction is still unknown.
The first R-gene cloned was the Hm1 gene from corn (Zea mays), which confers resistance to specific races of the fungal pathogen Cochliobolus carbonum (Johal et al., 1992, Science 258:985-987). Hm1 encodes a reductase that detoxifies a toxin produced by the pathogen. Next to be cloned was the Pto gene from tomato (Lycopersicon pimpinellifollium) (Martinet al., 1993, Science 262:1432-1436; U.S. Pat. No. 5,648,599). Pto encodes a serine-threonine protein kinase that confers resistance in tomato to strains of the bacterial pathogen Pseudomonas syringae pv. tomato that express the avrPto avirulence gene. Taking center stage now are R-genes that encode proteins containing leucine-rich-repeats (LRRs) (Jones and Jones, 1997, Adv. Bot. Res. Incorp. Adv. Plant Pathol. 24:89-167). Two classes of membrane anchored proteins with extracellular LRRs have been identified. One subclass includes R-gene products that lack a cytoplasmic serine/threonine kinase domain such as the tomato Cf-9 gene for resistance to the fungus Cladosporium fulvum (Jones et al., 1994, Science 266:789-793, WO95/18230), and the other subclass includes an R-gene product with a cytoplasmic serine/threonine kinase domain, the rice Xa-21 gene for resistance to the bacterial pathogen Xanthomonas oryzae (Song et al., 1995, Science 270:1804-1806; U.S. Pat. No. 5,859,339). The largest class of R-genes includes those encoding proteins with cytoplasmic LRRs such as the Arabidopsis R-genes RPS2 (Bent et al., 1994, Science 265:1856-1860; Mindrinos et al., 1994, Cell 78:1089-1099) and RPM1 (Grant et al., 1995, Science 269:843-846). These R-proteins also possess a putative nucleotide binding site (NBS), and either a leucine zipper (LZ) motif or a sequence homologous to the Toll/Interleukin-1receptor (TIR). Table 1 has been reproduced in part from Baker et al. (1997, Science 276:726-733) as a concise summary of classes of R-genes cloned to date and examples of cloned genes within each class.
Nucleotide binding sites (NBS) are found in many families of proteins that are critical for fundamental eukaryotic cellular functions such as cell growth, differentiation, cytoskeletal organization, vesicle transport, and defense. Key examples include the RAS group, adenosine triphosphatases, elongation factors, and heterotrimeric GTP binding proteins called G-proteins (Saraste et al., 1990, Trends in Biochem. Science 15:430). These proteins have in common the ability to bind ATP or GTP (Traut, 1994, Eur. J. Biochem. 229:9-19).
It has long been hypothesized that the rice blast system represented a classical gene-for-gene system as defined by H. H. Flor (Flor, 1971, Annu. Rev. Phytopathol. 19:125-143). Genetic analyses needed to identify AVR-genes in the rice blast pathogen, Magnaporthe grisea, has been hampered by the low fertility that typifies M. grisea field isolates that infect rice. Genetic crosses between poorly fertile M. grisea rice pathogens and highly fertile M. grisea pathogens of other grasses (such as weeping lovegrass, Eragrostis curvula, and finger millet, Eleusine coracana) have provided laboratory strains of the fungus with the level of sexual fertility required for identifying AVR-genes (Valent et al., 1991, Genetics 127:87-101). Rare fertile rice pathogens have since allowed demonstration of a one-to-one genetic or functional correspondence between blast fungus AVR-genes and particular rice R-genes (Silue et al., 1992, Phytopathology 82:577-580).
Interest in the rice blast pathosystem is keen because rice blast disease, caused world-wide by the fungal pathogen Magnaporthe grisea (Hebert) Barr (anamorph Pyricularia grisea Sacc.), continues as the most explosive and potentially damaging disease of the rice crop despite decades of research towards its control. Manipulation of blast resistance genes remains one of the primary targets in all rice breeding programs, as fungal populations evolve to defeat deployed resistance strategies (See The Rice Blast Disease, 1994, ed. Zeigler, Leong and Teng, CAB International, Wallingford).
Commercial fungicide usage to supplement genetic control strategies began around 1915 when rice farmers used inorganic copper-based fungicides (Chapter 29 in The Rice Blast Disease, 1994, ed. Zeigler, Leong and Teng, CAB International, Wallingford). The fungicides used to control blast disease have changed through time, with some compounds, such as the organomercurials used in the 1950s, causing major environmental damage. The control of rice blast with fungicides currently represents a cost of more than $500 million per year to farmers. This expense for blast control is the largest segment of the world rice fungicide market, which totaled $752 million in 1998 (Wood Mackenzie). Expectations are that the disease problems will intensify as the world rice requirements increase by an estimated 1.7% annually between 1990 and 2025 (See The Rice Blast Disease, 1994, ed. Zeigler, Leong and Teng, CAB International, Wallingford). This estimated need for an additional 13 million tons of rough rice per year to feed the growing population must come from intensification of production on decreasing available land. Rice blast disease is favored by agronomic production practices aimed at high yields, and thus the disease will continue, and most likely increase, as a constraint to rice crop yields unless durable genetic resistance against rice blast disease can be engineered into rice. This invention is among one of the first rice blast resistance genes to be cloned, represents a critical first step to the long term goal of engineering durable genetic resistance to rice blast disease.
The fungus M. grisea has a large host range including species of different tribes within the grass family, Triticeae (e.g., wheat), Oryzeae (e.g., rice), Clorideae (e.g., finger millet), Paniceae (e.g., pearl millet), Andropogoneae (e.g., Sorghum) and Maydeae (e.g., maize). Molecular analyses have now defined 8 host species-specific subpopulations of M. grisea, each with a restricted set of host species specificities (Reviewed by Valent, 1997, The Mycota V, Plant Relationships, Carroll/Tudzynski, eds., Springer-Verlag Berlin Heidelberg pp 37-54). Table 2 gives a current view of pathogen subpopulations according to mitochondrial DNA (mtDNA) type. This view is strongly supported by separate analyses of ribosomal DNA (rDNA) polymorphisms (including both Restriction Fragment Length Polymorphism (RFLP) and Internal Transcribed Spacer (ITS) sequences) and of polymorphisms in both repetitive DNAs and single copy sequences.
The pathogens of rice, wheat, finger millet, barley and corn (mtDNA types Ia-e) appear closely related, while pathogens of Digitaria spp. and Pennisetum spp. (mtDNA types II-IV) are highly divergent from the previous groups and from each other. However, M. grisea strains throughout this broad host range can cause significant crop damage. This pathogen has been shown to be the main cause of yield loss of finger millet (Eleusine coracana) in Africa, while infections in wheat (Triticum aestivum; Urashima et al., 1993, Plant Disease 77:1211-1216) and pearl millet (Pennisetum glaucum; Hanna et al., 1989, J. Heredity 80:145-147), although less widespread, can be severe under humid weather conditions. The disease has been documented on barley and corn (See refs. In Urashima et al., 1993, Plant Disease 77:1211-1216).
Knowledge of pathogenicity and host specificity for plant pathogenic fungi is not as advanced as for bacterial and viral pathogens, and likewise, less is known about the molecular basis of resistance in cereal crop plants than in dicot crops or in dicot model systems such as Arabidopsis (Baker et al., 1997, Science 276:726-733). Sasaki reported the first results on the inheritance of resistance to rice blast disease from studies begun in Japan in 1917 (Sasaki, 1922, Japanese Genetics, Japan 1, 81-85).
Since this time, over 30 R-genes have been defined through extensive genetic analysis worldwide, and many of these blast resistance genes have been mapped to rice chromosomes (See Refs. In Takahashi, 1965, The Rice Blast Disease, Johns Hopkins Press, Baltimore, 303-329; Causse et al., 1994, Genetics 138:1251-1274). These R-genes include 20 major resistance genes and 10 putative quantitative trait loci (QTLs). Kiyosawa has described 13 major resistance genes with 9 of these genes found as multiple alleles at 3 loci; at the Pi-k locus on chromosome 11, 2 at the Pi-z locus on chromosome 6 and 2 at the Pi-ta locus on chromosome 12 (Kiyosawa, 1984, Rice Genetics Newsletter 1:95-97). Recent studies in Japan (Ise, 1992, International Rice Research Newsletter 17:8-9) and at the International Rice Research Institiute (IRRI) (Mackill et al., 1992, Phytopathology 82:746-749) have produced near isogenic rice lines (NILs) for use as xe2x80x9cdifferentialxe2x80x9d rice varieties for determining which resistance genes are effective in controlling individual strains of the fungus. The IRRI NILs, which provide indica differentials for the blast fungus populations in tropical regions, have been analyzed for genetic relationships between their resistance genes and those present in Kiyosawa Differentials (Inukai et al., 1994, Phytopathology 84:1278-1283).
Molecular markers (or xe2x80x9ctagsxe2x80x9d) tightly linked to R-genes have utility for efficient introgression and manipulation of those R-genes in breeding programs. By comparing genotypic patterns of near-isogenic lines, their donors, and their recurrent parents, Yu et al. (1987, Phytopathology 77:323-326) were able to identify five restriction fragment length polymorphic (RFLP) markers linked to three blast resistance genes and to map them to rice chromosomes using segregating populations. RFLP markers linked to the R-genes have been reported (Yu et al., 1991, Theor Appl Genet 81:471-476). Molecular cloning of agronomically important R-genes represents a further advance to the ability of researchers to combine R-genes with other input and output traits in key crop varieties.
In the course of the above mentioned investigations on the inheritance of resistance, Sasaki discovered physiological races of the rice blast pathogen by observing that different field isolates of the blast fungus vary in their ability to cause disease on different varieties of rice (Sasaki, 1922, Journal of Plant Protection 9:631-644; Sasaki, 1923, Journal of Plant Protection 10:1-10). Instability, or xe2x80x9cbreaking downxe2x80x9d under field conditions, of major R-gene resistance to the rice blast fungus has resulted in identification of numerous races, or pathotypes, defined according to virulence spectra on differential rice varieties (Chapters 13 and 16 in The Rice Blast Disease, 1994, ed. Zeigler, Leong and Teng, CAB International, Wallingford). Pathogen populations are dynamic in response to deployment of a new resistance gene, sometimes resulting in new races that overcome the resistance gene within one or two years after deployment in the field.
Researchers have not yet characterized in depth an avirulence gene/R-gene pair in the rice blast pathosystem.
U.S. Pat. No. 5,648,599, issued to Tanksley and Martin on Jul. 15, 1997, describes an isolated gene fragment from tomato which encodes the Pto serine/threonine kinase, conferring disease resistance to plants by responding to an avirulence gene in a bacterial plant pathogen.
WO 95/28423, which published on Oct. 26, 1995, describes resistance due to the Pseudomonas syringae RPS2 gene family, primers, probes and detection methods. This published international application includes broad claims to genes encoding proteins with particular NH2-terminal motifs, NBS motifs and leucine rich repeats for protecting plants against pathogens. There are some unique features of the Pi-ta protein. The Pi-ta gene product has a unique amino terminus, lacking either the potential leucine zipper motif of the RPS2 gene-product subfamily (Bent et al., 1994, Science 265:1856-1860; Mindrinos et al., 1994, Cell 78:1089-1099) or the Toll/Interleukin-1 receptor homology encoded by the N gene subfamily (Whitman et al., 1994, Cell 78:1101-1115). Most importantly, the carboxy terminal portion of the Pi-ta gene product is leucine rich, but it does not fit the consensus sequences for leucine-rich repeats reported for R-gene products (Jones and Jones, 1997, Adv. Bot. Res. Incorp. Adv. Plant Pathol. 24:89-167).
U.S. Pat. No. 5,571,706, issued to Baker et al. on Nov. 5, 1996, covers plant virus resistance conferred by the N gene.
U.S. Pat. No. 5,859,351, issued to Staskawicz et al. on Jan. 12, 1999, describes the PRF protein and nucleic acid sequence, which is involved in disease resistance in tomato.
U.S. Pat. No. 5,859,339, issued to Ronald et al. on Jan. 12, 1999, describes the first resistance gene cloned from rice, Xa-21, which encodes an integral membrane protein with both LRR and serine/threonine kinase domains, and confers resistance in rice to bacterial blight.
WO 91/15585 which published on Oct. 17, 1991 and U.S. Pat. No. 5,866,776 issued to de Wit et al. on Feb. 2, 1999 describe a method for the protection of plants against pathogens using a combination of a pathogen avirulence gene and a corresponding plant resistance gene.
U.S. Pat. No. 5,674,993 (""993 patent), issued to Kawasaki et al. on Oct. 7, 1997, describes nucleic acid markers that co-segregate with the rice blast resistance genes Pi-b, Pi-ta and Pi-ta2 and the suggestion that rice blast resistance genes could be isolated and cloned by using these nucleic acid markers. However, no nucleotide sequences are provided for any rice blast resistance genes in the ""993 patent. It should be noted that a putative sequence for the Pi-b rice blast resistance gene is now available in Genbank (accession number AB013448).
In addition, Kawasaki et al. have also published two papers. The first paper, Rybka et al., MPMI, 10(4):517-524 (1997), is entitled xe2x80x9cHigh Resolution Mapping of the Indica-Derived Rice Blast Resistance Genes. II. Pi-ta2 and Pi-ta and a Consideration of Their Origin.xe2x80x9d The sequence for the RAPD primer that is set forth at the top of column 2 on page 519 is not the same as the RAPD primer set forth in SEQ ID NO:2 in the ""993 patent. The sequence for the primer in the paper is TCCCCAGCCA (SEQ ID NO:75). The sequence for the primer in the ""993 patent is TCGCCAGCCA (SEQ ID NO:76). It is not clear which sequence is correct. Notwithstanding this, it is clear that this paper does not set forth any nucleotide sequences for any rice blast genes. The second paper is Nakamura et al., Mol. Gen. Genet. 254:611-62 (1997). This paper describes the construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta2 using a rice BAC library. Again, no nucleotide sequence for any rice blast genes is disclosed.
Thus, it is believed that no one hereto has cloned and sequenced a Pi-ta resistance gene from rice with demonstrated utility in controlling Magnaporthe grisea that would have significant value as crop protection tools for other graminaceous crops as well. Accordingly, the invention described herein represents the first molecular characterization of a Pi-ta avirulence gene/R-gene pair in the rice blast pathosystem.
This invention relates to an isolated nucleic acid fragment which confers a Pi-ta resistance gene-mediated defense response against diseases caused by fungal pathogens wherein said nucleic acid fragment corresponds substantially to the nucleotide sequence set forth in SEQ ID NOS:1 or 68 or a functionally equivalent subfragment thereof.
In another aspect this invention concerns genes comprising isolated nucleic acid which confers a Pi-ta resistance gene-mediated defense response against diseases caused by fungal pathogens wherein said nucleic acid fragment corresponds substantially to the nucleotide sequence set forth in SEQ ID NOS:1 or 68 or a functionally equivalent subfragment thereof wherein these fragments are operably linked to suitable regulatory sequences.
In still another aspect, this invention concerns a method of conferring Pi-ta resistance gene-mediated defense response in plants against disease caused by a fungal pathogen which comprises introducing into the plant a gene comprising isolated nucleic acid which confers a Pi-ta resistance gene-mediated defense response against diseases caused by fungal pathogens wherein said nucleic acid fragment corresponds substantially to the nucleotide sequence set forth in SEQ ID NOS:1 or 68 or a functionally equivalent subfragment thereof wherein these fragments are operably linked to suitable regulatory sequences.
In an even further aspect, this invention concerns a method of conferring a resistance gene-mediated defense response in plants at the site of infection by a fungal pathogen, comprising transforming the plant with a recombinant expression construct which comprises (1) an isolated nucleic acid fragment which confers a Pi-ta resistance gene-mediated defense response against diseases caused by fungal pathogens wherein said nucleic fragment corresponds substantially to the nucleotide sequence set forth in SEQ ID NOS:1 or 68 or a functionally equivalent subfragment thereof and (2) an AVR-Pita isolated nucleic acid fragment corresponding substantially to the nucleotide sequence set forth in SEQ ID NO:5 or 7 or a functionally equivalent subfragment thereof, wherein said nucleic acid fragments are operably linked to regulatory sequences and further wherein said construct confers a resistance gene-mediated defense response in plants against diseases caused by a fungal pathogen at the site of infection.
This invention also concerns a recombinant expression construct which confers a resistance gene-mediated defense response in plants against diseases caused by a fungal pathogen at the site of infection which comprises (1) an isolated nucleic acid fragment which confers a Pi-ta resistance gene-mediated defense response against diseases caused by fungal pathogens wherein said gene fragment corresponds substantially to the nucleotide sequence set forth in SEQ ID NOS:1 or 68 or a functionally equivalent subfragment thereof and (2) an AVR-Pita isolated nucleic acid fragment corresponding substantially to the nucleotide sequence set forth in SEQ ID NO:5 or 7 or a functionally equivalent subfragment thereof, wherein said nucleic acid fragments are operably linked to regulatory sequences.
Also of interest are plants transformed with such a gene or recombinant expression construct and seeds obtained from such plants.
The fungal strain O-137 has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and bears the following designation, accession number and date of deposit. Fungal strain G-213, a pathogen isolated from Digitaria smutsii in Japan, was obtained from the collection of Jean Loup Notteghem, Laboratoire de phytopathologie, Institut de Recherches Agronomiques Tropicales et des Cultures Vivrieres, Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement (CIRAD), BP 5035, 34032 Montpellier Cedex 1, France, and is available under the name JP34. This strain has also been deposited with the ATCC.
The invention can be more fully understood from the following detailed description and the Sequence Descriptions which form a part of this application. The Sequence Descriptions contain the three letter codes for amino acids as defined in 37 C.F.R. 1.822 which are incorporated herein by reference. The nucleotide sequences read from 5xe2x80x2 to 3xe2x80x2.
SEQ ID NO:1 is the 5757 nucleotide sequence of the genomic clone of the Pi-ta gene from Oryza sativa variety Yashiro-mochi.
SEQ ID NO:2 is the predicted protein sequence encoded by the Pi-ta gene set forth in SEQ ID NO:1.
SEQ ID NO:3 is the 5113 nucleotide sequence of the genomic clone of the pi-ta gene from susceptible Oryza sativa variety Tsuyuake.
SEQ ID NO:4 is the predicted protein sequence encoded by the pi-ta gene set forth in SEQ ID NO:3.
SEQ ID NO:5 is the 672 nucleotide coding sequence of an AVR-Pita (formerly called AVR2-YAMO) cDNA from M. grisea rice pathogen O-137.
SEQ ID NO:6 is the predicted protein sequence encoded by the sequence set forth in SEQ ID NO:5.
SEQ ID NO:7 is the 675 nucleotide coding sequence of an AVR-Pita cDNA from M. grisea pathogen G-213 that infects Digitaria species.
SEQ ID NO:8 is the predicted protein sequence encoded by the sequence set forth in SEQ ID NO:7.
SEQ ID NOS:9-24 set forth the sequences of oligonucleotide (RAPD) primers used in the genetic mapping and cloning of the Pi-ta region.
SEQ ID NOS:25-31 are oligonucleotide primers used in PCR amplification of BAC insert ends.
SEQ ID NOS:32 and 33 are PCR primers used to verify the location of RAPD marker SP4B9.
SEQ ID NOS:34-36 are partial sequences obtained from BAC clone RB142E8 which contains a single copy of the Pi-ta candidate gene PRG2.
SEQ ID NOS:37-64 are sequencing primers used in the full sequencing of the genomic clone for Pi-ta (PRG2) set forth in SEQ ID NO:1.
SEQ ID NO:65 is an oligonucleotide primer used to obtain a Pi-ta nucleic acid fragment.
SEQ ID NOS:66 and 67 are PCR primers used to amplify AVR-Pita176 an AVR-Pita nucleic acid fragment that directly encodes the putative mature protease.
SEQ ID NO:68 is the 5222 nucleotide sequence of an EcoRI-HindIII fragment that contains 2425 bp of the native Pi-ta promoter (nucleotides 1 to 2425) and Pi-ta cDNA (nucleotides 2426-5212).
SEQ ID NOS:69 and 70 are oligonucleotide primers used to generate a linker fragment in constructing pML135.
SEQ ID NOS:71 and 72 are PCR primers used to amplify the Adh1-6 intron from maize genomic DNA.
SEQ ID NOS:73 and 74 are PCR primers used to amplify the AVR-Pita nucleic acid fragment (SEQ ID NO:5) in the process of constructing pAVR3.